Kir3 or GIRK (G protein gated inwardly rectifying K+) channels are activated by the vagus nerve to control heart rate. They are critical determinants of heart rate variability (HRV), an index of cardiac health, endowing the heart with the adaptability it needs to make rapid adjustments in heart rate. GIRK channels are attractive drug targets against atrial fibrillation (AF), the most common arrhythmia whose prevalence increases with age with an increased risk of mortality, stroke and myocardial infarction. The lack of specificity of the current antiarrhythmics used poses significant risk for ventricular side effects. This makes rather attractive targets expressed predominantly in the atria. Overactivity of cardiac GIRK channels has been implicated under the oxidative stress conditions characteristic of aging through a dysregulation of Protein Kinase C (PKC) enzymes, such as the increase in activity of the novel PKCe. Yet, even though full inhibitors of GIRK activity could reverse AF, they would also inhibit HRV, a side effect detrimental to cardiac health. Thus, the need for specific partial inhibitors that reverse the PKC-mediated channel activation but do not inhibit the vital functions of the channel is an unmet medical need. In this proposal, we investigate the mechanism by which PKC-dependent phosphorylation affects activity and show that it allosterically affects the interactions of the channel with PIP2, the master regulator of membrane protein function. We identify one phosphorylation site used by PKCe to stimulate channel activity and propose to determine all the sites involved and identify the gates they allosterically couple with to cause channel activation. In parallel, we have developed powerful structural computational models that allow us to test the action of small molecule inhibitors, which also allosterically control distinct channel gates via PIP2. In this proposal, we aim to set the stage in coupling the molecular insights of small molecule regulators of activity to specifically reverse the PKC-mediated overstimulation of channel activity. Our small molecule inhibitors will be tested in transgenic models of PKC-mediated AF with the goal to dial down the aberrant activity enough to correct the AF problem without compromising cardiac health.